The present disclosure, according to one embodiment, relates to compositions and methods for detecting molecules in a sample using a synthetic receptor core molecule.
A common objective when developing synthetic receptors is high binding affinity and specificity for an analyte. This often may be challenging when targeting a complex analyte in a competitive crude medium such as a biological specimen (e.g., serum, urine, or saliva. See S. L. Tobey & E. V. Anslyn, Org. Lett. 5:2029-31(2003); Z. Zhong & E. V. Anslyn, Angew. Chem. 115:3113-16 (2003).
One example of an analyte for a synthetic receptor is heparin. Heparin is a heterogeneous mixture of diverse chain lengths consisting of repeating copolymers of 1→4 linked iduronic acid and glucosamine residues in a semi-random order. For a biopolymer, heparin has a very high anionic charge to mass ratio, as a result of numerous sulphate and carboxylate functionalities in the biopolymer chain.
In general, two forms of heparin are in clinical use, unfractionated heparin (UFH) with a molecular weight range of from about 3,000 to about 30,000 Da, and low-molecular-weight heparin (LMWH) with a mean molecular weight of about 5,000 Da. During surgery, and in post-operative therapy, heparin concentration and activity is monitored to prevent complications such as hemorrhaging.
Heparin's anticoagulant activity occurs by binding to antithrombin III, a naturally occurring protease inhibitor, accelerating the rate of inhibition of coagulation proteases factor Xa and thrombin by antithrombin III. Clinically administered heparin binds to its natural substrate antithrombin III primarily through cationic ion-pairing interactions with the sulphates and carboxylates. Similarly, the cationic protein protamine is another ligand for heparin.
Current methods for heparin quantification employ the Activated Clotting Time (ACT), Activated Partial Thromboplastin Time (aPTT), chromogenic anti-factor Xa assay, electrochemical and piezoelectric assays, and complexation with protamine. Nonclinically, heparin also has been quantified using an engineered GST-fusion protein containing three hyaluronan-binding domains from a heparin binding protein, but it has not been employed clinically. J. L. Cai, et al., Anal. Biochem. 326:33-4 (2004). These methods may be problematic, however, as they may be difficult, inaccurate, costly, and not amenable to clinical settings.